The overall goal of this project is to identify factors that regulate the function of the TAR DNA binding protein (TDP-43). TDP-43 is the main disease protein accumulating in cytoplasmic inclusion bodies in amyotrophic lateral sclerosis (ALS) and front temporal lobar degeneration (FTLD), two major forms of neurodegeneration. TDP-43 is predominantly nuclear and mediates RNA processing. TDP-43 aggregation is accompanied by the loss of nuclear localization, suggesting that lack of at least one of the protein's nuclear functions is associated with neurodegeneration. In addition, the importance of TDP-43 in disease is highlighted by more than 40 dominant mutations found in familial and sporadic forms of ALS-FTLD. The role of TDP-43 in disease, however, remains unknown. Little information exists on factors that regulate TDP-43 and whether posttranslational modifications (PTMs) affect TDP-43 function. Our preliminary search identified specific TDP- 43 residues that undergo phosphorylation under physiological conditions and multiple other predicted phosphosites. Based on their positions, the reported and predicted phosphosites may profoundly affect key TDP-43 processes, e.g. cellular trafficking and RNA interactions. In addition, almost half of the patient-linked TDP-43 mutations potentially increase protein phosphorylation. We hypothesize that TDP-43 is regulated by phosphorylation and that several disease-associated mutations change the phosphorylation profile with deleterious consequences on protein function. We propose to (aim 1) test the effect of phosphorylation on protein function, including the novel phosphosites identified through our proposed analyses; (aim 2) determine if protein activity and solubility are affected by the patient-linked mutations that change the phosphorylation profile; and (aim 3) characterize the function of TDP-43 phosphorylation in stress granule localization. Cell culture models will be used to identify phosphosites by mass spectrometry. The role of phosphorylation on protein function will be probed using site- directed mutants in well-established cellular assays to monitor TDP-43 activity and solubility. The current lack of information regarding TDP-43 physiological phosphorylation and that associated with patient-derived mutants underscores the importance of our studies to understand protein function and its role in disease. Our long-term goal is to identify the factors and cellular pathways that regulate TDP-43 and to contribute in the elucidation of processes that cause neurodegeneration. The candidate has a long-standing interest in the characterization of proteins that mediate RNA processing, particularly the investigation of TDP-43 function. The NINDS Faculty Development Award to Promote Diversity in Neuroscience Research will provide structured training and focus on neurological disease mechanisms. As a recently established independent faculty in the Edward A. Doisy Department of Biochemistry and Molecular Biology at Saint Louis University, the candidate is committed to continuing the elucidation of molecular mechanisms associated with neurodegeneration. An outstanding team of mentors from the Department of Biochemistry and Molecular Biology and the Hope Center for Neurological Disorders at Washington University, St. Louis, will monitor and evaluate the research progress and the achievement of the candidate's career goals. As part of the career development program, the candidate will actively participate in the Hope Center for Neurological Disorders and the Hope Center Program on Protein Aggregation & Neurodegeneration at Washington University, St. Louis. This will greatly contribute to the candidate's preparation in the field of neurodegeneration and increase the translational potential of the candidate's research.